Method for improving the stability of purified Factor VIII after reconstitution

ABSTRACT

The present disclosure relates to a method for increasing the stability of a Factor VIII molecule after purification, lyophilization and reconstitution, comprising preventing proteolytic cleavage of the Factor VIII molecule into a first fragment comprising essentially the A1 domain and the A2 domain and a second fragment comprising essentially the A3 domain, the C1 domain and the C2 domain throughout manufacturing the Factor VIII molecule. The disclosure further pertains to a method for improving the bioavailability of Factor VIII after intravenous and non-intravenous injection.

This is a continuation of application Ser. No. 16/701,468, filed Dec. 3,2019, now U.S. Pat. No. 10,881,717, issued Jan. 5, 2021, which is acontinuation application of application Ser. No. 15/899,510, filed Feb.20, 2018, now U.S. Pat. No. 10,537,616, issued Jan. 21, 2020, which is acontinuation application of application Ser. No. 15/156,744, filed May17, 2016, now U.S. Pat. No. 9,956,269, issued May 1, 2018 which is acontinuation application of application Ser. No. 14/351,513, now U.S.Pat. No. 9,394,353, issued Jul. 19, 2016, having a 35 U.S.C. § 371(c)date of Apr. 11, 2014, which is the national stage entry ofInternational Application No. PCT/EP2012/070701, filed Oct. 18, 2012,which claims priority to U.S. Provisional Application No. 61/548,601,filed Oct. 18, 2011, and also claims priority to European PatentApplication No. 11185651.4, filed Oct. 18, 2011, all of which areincorporated herein by reference.

The instant application contains a Sequence Listing that is submittedherewith electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created Nov. 27, 2020, isnamed SequenceListing.txt and is 56,086 bytes in size. Please insert theSequence Listing filed herewith into the application before the claims.In connection with said Sequence Listing, the undersigned hereby statesthat:

1. The sequence listing, filed herewith in accordance with 37 C.F.R. §§1.821-1.825, does not include new matter; and

2. The attached ASCII-format sequence listing serves as both a writtencopy and the computer readable copy of the Sequence Listing; thus, thecontents of the written and computer readable copies of the SequenceListing are the same.

The present invention relates to a method for increasing the stabilityof a Factor VIII molecule after purification, lyophilization andreconstitution, comprising preventing proteolytic cleavage of the FactorVIII molecule into a first fragment comprising essentially the A1 domainand the A2 domain and a second fragment comprising essentially the A3domain, the C1 domain and the C2 domain throughout manufacturing of theFactor VIII molecule. The invention further pertains to a method forimproving the bioavailability of Factor VIII after intravenous andnon-intravenous injection.

BACKGROUND OF THE INVENTION

Classic hemophilia or hemophilia A is an inherited bleeding disorder. Itresults from a chromosome X-linked deficiency of blood coagulationFactor VIII, and affects almost exclusively males with an incidence ofbetween one and two individuals per 10,000. The X-chromosome defect istransmitted by female carriers who are not themselves hemophiliacs. Theclinical manifestation of hemophilia A is an increased bleedingtendency. Before treatment with Factor VIII concentrates was introducedthe mean life span for a person with severe hemophilia was less than 20years. The use of concentrates of Factor VIII from plasma hasconsiderably improved the situation for the hemophilia patientsincreasing the mean life span extensively, giving most of them thepossibility to live a more or less normal life. However, there have beencertain problems with the plasma derived concentrates and their use, themost serious of which have been the transmission of viruses. So far,viruses causing AIDS, hepatitis B, and non-A non-B hepatitis have hitthe population seriously. Since then different virus inactivationmethods and new highly purified Factor VIII concentrates have recentlybeen developed which established a very high safety standard also forplasma derived Factor VIII.

Several recombinant and plasma-derived, therapeutic polypeptides, e.g.blood coagulation factors, are commercially available for therapeuticand prophylactic use in humans. FVIII is a blood plasma glycoprotein ofup to about 280 kDa molecular mass, produced in the liver of mammals. Itis a critical component of the cascade of coagulation reactions thatlead to blood clotting. Within this cascade is a step in which factorIXa (FIXa), in conjunction with activated factor VIII (FVIIIa), convertsfactor X (FX) to an activated form, FXa. FVIIIa acts as a cofactor atthis step, being required together with calcium ions and phospholipidsfor maximizing the activity of FIXa.

An important advance in the treatment of hemophilia A has been theisolation of cDNA clones encoding the complete 2,351 amino acid sequenceof human FVIII (U.S. Pat. No. 4,757,006) and the provision of the humanFVIII gene DNA sequence and recombinant methods for its production).

Factor VIII is synthesized as a single polypeptide chain with amolecular weight of about 280 kDa. The amino-terminal signal peptide isremoved upon translocation of factor VIII into the endoplasmaticreticulum, and the mature (i.e. after the cleavage of the signalpeptide) native Factor VIII molecule is then proteolytically cleavedafter amino acid residues 1313 and 1648 in the course of its secretion.This results in the release of a heterodimer which consists of aC-terminal light chain of about 80 kDa in a metal ion-dependentassociation with an about 160-200 kDa N-terminal heavy chain fragment.(See review by Kaufman, Transfusion Med. Revs. 6:235 (1992)).

Physiological activation of the heterodimer occurs through proteolyticcleavage of the protein chains by thrombin. Thrombin cleaves the heavychain to a 90 kDa protein, and then to 54 kDa and 44 kDa fragments.Thrombin also cleaves the 80 kDa light chain to a 72 kDa protein. It isthe latter protein, and the two heavy chain fragments (54 kDa and 44 kDaabove), held together by calcium ions, that constitute active FVIII.Inactivation occurs when the 44 kDa A2 heavy chain fragment dissociatesfrom the molecule or when the 72 kDa and 54 kDa proteins are furthercleaved by thrombin, activated protein C or FXa. In plasma, FVIII isstabilized by association with a 50-fold molar excess of VWF protein(“VWF”), which appears to inhibit proteolytic destruction of FVIII asdescribed above.

The amino acid sequence of FVIII is organized into three structuraldomains: a triplicated A domain of 330 amino acids, a single B domain of980 amino acids, and a duplicated C domain of 150 amino acids. The Bdomain has no homology to other proteins and provides 18 of the 25potential asparagine (N)-linked glycosylation sites of this protein. TheB domain has apparently no function in coagulation and can be deletedwith the B-domain deleted FVIII molecule still having procoagulatoryactivity.

The Factor VIII products on the market are currently presented as alyophilized formulation of Factor VIII either produced by recombinanttechnology or purified from pooled plasma. The lyophilized product isreconstituted prior to administration. Once reconstituted, shelf-life ofthe Factor VIII is relatively short. Factor VIII is a relativelyunstable protein, particularly in aqueous solutions. Stabilizationduring manufacturing and storage by complexing with other plasmaproteins, particularly von Willebrand factor (vWF) and albumin, has beendescribed. See, for example, U.S. Pat. No. 6,228,613. U.S. Pat. No.5,565,427 discloses a stabilized formulation of Factor VIII comprisingan amino acid or one of its salts or homologues and a detergent or anorganic polymer such as polyethyleneglycol. U.S. Pat. No. 5,605,884discloses stabilized formulations of Factor VIII in high ionic strengthmedia based on histidine buffer in the presence of calcium chloride anda high concentration of sodium chloride or potassium chloride. Suchcompositions were shown to improve significantly the stability of FactorVIII in aqueous form following reconstitution. The importance of calciumions in the formulations of Factor VIII is generally recognized.According to U.S. Pat. No. 6,599,724, the presence of other divalentcations, namely Cu²⁺ and Zn²⁺, optionally in the presence of Ca²⁺ ionsor Mn²⁺ ions improves the stability of Factor VIII. Also WO 2011/027152A1 describes stable aqueous Factor VIII compositions comprising variousadditives.

In view of the short shelf life of Factor VIII after reconstitution of alyophilisate, there is a need for methods to increase the stability ofreconstituted Factor VIII in aqueous solution. To provide a purifiedFVIII preparation with increased stability in the liquid phase isdesirable for different reasons. First of all, it is of advantage tohave a sufficient time span at ambient temperature to supportmanufacturing of the purified FVIII product at ambient temperature. Inparticular, the filling step necessitates some storage of a liquid bulkto increase flexibility in manufacturing. Secondly, an increasedstability of the liquid purified FVIII would be of advantage forphysician and patient if the product could not be applied directly afterreconstitution. And finally, the use of FVIII under continuous infusionconditions e.g. upon surgery in hospitalized patients is depending on apreferably high product stability after reconstitution (Takedani H.,Haemophilia 2010, 16: 740-746). A FVIII molecule with increasedstability would also be an advantage for development of a FVIIIpreparation suitable for long term storage under liquid conditions.

The inventors of this application surprisingly found that the stabilityof purified Factor VIII after reconstitution of a lyophilisate issubstantially enhanced in single-chain Factor VIII constructs. Suchconstructs can be obtained by preventing the proteolytic cleavage whichtypically occurs in the Golgi compartment prior to secretion of FactorVIII. The single-chain constructs exhibit a better stability in solutionafter purification and/or a better bioavailability upon subcutaneousadministration.

SUMMARY OF THE INVENTION

In a first aspect the present invention relates to a method forincreasing the stability of a Factor VIII molecule after purification,lyophilization and reconstitution, comprising preventing proteolyticcleavage of the Factor VIII molecule into a first fragment comprisingessentially the A1 domain and the A2 domain and a second fragmentcomprising essentially the A3 domain, the 01 domain and the C2 domain.

The first aspect encompasses a method for increasing the stability of aFactor VIII molecule after purification, lyophilization andreconstitution, comprising preventing proteolytic cleavage of the FactorVIII molecule into a first fragment comprising essentially the A1 domainand the A2 domain and a second fragment comprising essentially the A3domain, the C1 domain and the C2 domain throughout manufacturing of theFactor VIII molecule.

The first aspect further encompasses a method for increasing thestability of a Factor VIII molecule after purification, lyophilizationand reconstitution, comprising preventing proteolytic cleavage of theFactor VIII molecule into a first fragment comprising essentially the A1domain and the A2 domain and a second fragment comprising essentiallythe A3 domain, the C1 domain and the C2 domain prior to the purificationof the Factor VIII molecule.

With regard to these methods according to the invention the terms“throughout manufacturing of the Factor VIII molecule” and “prior to thepurification of the Factor VIII molecule” are intended to mean that themethods of the invention prevent the cleavage of Factor VIII into afirst fragment comprising essentially the A1 domain and the A2 domainand a second fragment comprising essentially the A3 domain, the C1domain and the C2 domain but the methods according to the invention donot prevent the activation cleavage of Factor VIII which may occur afteradministration of the reconstituted Factor VIII molecule. The FactorVIII molecules generated by the methods of the invention can still beactivated by thrombin which cleaves the Factor VIII molecule after Arg372, Arg 740 and Arg 1689.

In a second aspect, the present invention relates to a method forincreasing the stability of a Factor VIII molecule after purification,lyophilization and reconstitution, comprising inactivating theproteolytic cleavage sites which are cleaved during secretion of saidFactor VIII molecule by the host cell expressing the Factor VIIImolecule except the cleavage site between the signal sequence and themature Factor VIII. Typically, at least 50% of the Factor VIII moleculesexpressed and secreted by the host cells are single-chain Factor VIIImolecules. Preferably, at least 60%, or at least 70%, or at least 80%,or at least 90%, or at least 95% of the Factor VIII molecules expressedand secreted by the host cells are single-chain Factor VIII molecules.

Preferably, the method comprises inactivating the proteolytic cleavagesite between Arg1648 and Glu1649 and, if present in the FVIII molecule,the proteolytic cleavage site between Arg1313 and Ala1314. Theinactivation of the proteolytic cleavage site may be effected bydeleting one or more residues of the protease recognition sequence. Forexample, inactivation step may comprise deleting at least Arg1648 fromthe Factor VIII sequence. In one embodiment, the inactivation stepcomprises deleting at least the amino acid sequence from Arg1313 toArg1648 from the Factor VIII sequence.

In another embodiment of the first aspect of the invention theinactivation of the proteolytic cleavage site is effected bysubstituting one or more amino acid residues forming the proteaserecognition sequence.

In yet another embodiment (concerning those FVIII variants which retainthe part of the B-domain comprising Arg1313) the method furthercomprises inactivating the proteolytic cleavage site between Arg1313 andAla1314 by deletion or substitution of one or more residues forming theprotease recognition sequence. In a particularly preferred embodiment,the method comprises deleting at least a portion from the Factor VIIIamino acid sequence which comprises both protease cleavage sites afterresidues Arg1313 and Arg1648.

It is further preferred that a first amino acid selected from the aminoacids at positions 741 to 1647 of the Factor VIII sequence is fused witha second amino acid selected from the amino acids at positions 1649 to1690 of the Factor VIII sequence, whereby the proteolytic cleavage sitebetween Arg1648 and Glu1649 and, if present in the FVIII molecule, thecleavage site between Arg1313 and Ala1314 is inactivated.

In another preferred embodiment the Factor VIII molecule stabilized inaccordance with the first or second aspect of the invention exhibits anincreased stability in aqueous solution. The loss of activity of themodified Factor VIII molecule, in aqueous solution, after storage for 7days at 25° C. is preferably less than 15%.

In another preferred embodiment the Factor VIII molecule stabilized inaccordance with the first or second aspect of the invention exhibits anincreased stability in aqueous solution after reconstitution.

In yet another preferred embodiment the Factor VIII molecule stabilizedin accordance with the first or second aspect of the invention exhibitsan increased bioavailability after non-intravenous injection, ascompared to the bioavailability of human wild type Factor VIII or ascompared to a B-domain deleted Factor VIII molecule where Asn745 isfused to Pro1640, administered at the same dose and in the same manner.In yet another preferred embodiment the Factor VIII molecule stabilizedin accordance with the first or second aspect of the invention exhibitsan increased bioavailability after non-intravenous injection, ascompared to the bioavailability of a B-domain deleted Factor VIIImolecule where Asn745 is fused to Pro1640, administered at the same doseand in the same manner. The bioavailability of the modified FVIII ispreferably increased by at least 25%, as compared to the bioavailabilityof human wild type Factor VIII or of a B-domain deleted Factor VIIImolecule where Asn745 is fused to Pro1640, each administered at the samedose and in the same manner. In another preferred embodiment thenon-intravenous injection is subcutaneous, transdermal or intramuscularinjection.

Another preferred embodiment is a method wherein (i) the Factor VIIIexhibits improved plasma half-life after intravenous administrationrelative to human wild type Factor VIII; preferably wherein the plasmahalf-life is improved by at least 40% relative to human wild type FactorVIII, or (ii) wherein the Factor VIII exhibits a longer time period forthe thrombin peak level as determined in a thrombin generation assayover time in hemophilia A mice to fall below 50 nM after intravenousadministration relative to human wild type Factor VIII; preferablywherein this time period is prolonged by at least 10 hours relative tohuman wild type Factor VIII, or (iii) wherein the Factor VIII retains ahigher activity as determined by a one-stage FVIII:C assay after havingbeen incubated for 4 days in human plasma at 37° C. relative to humanwild type Factor VIII after having been incubated for 4 days in humanplasma at 37° C.; preferably wherein the retained activity of the FactorVIII is at least 10% higher relative to that of a human wild type FactorVIII after having been incubated for 4 days in human plasma at 37° C.

The methods may further comprise the steps of

(i) providing a nucleic acid encoding a modified Factor VIII molecule inwhich the proteolytic cleavage sites between Arg1648 and Glu1649, andbetween Arg1313 and Ala1314, are inactivated,

(ii) transforming a host cell with said nucleic acid,

(iii) culturing the transformed host cell under conditions such that themodified Factor VIII molecule is expressed, and

(iv) recovering the modified Factor VIII molecule from the host cells orfrom the culture medium.

In another aspect, the present invention relates to a method forimproving the bioavailability of a Factor VIII molecule afternon-intravenous administration, comprising inactivating the proteolyticcleavage site between Arg1648 and Glu1649 and, if present in the FVIIImolecule, the proteolytic cleavage site between Arg1313 and Ala1314.Preferably, the non-intravenous injection is subcutaneous injection. Thebioavailability after subcutaneous injection is preferably increased byat least 25% as compared to that of human wild type Factor VIII or of aB-domain deleted Factor VIII molecule where Asn745 is fused to Pro1640,each administered at the same dose and in the same manner.

In another aspect, the present invention relates to a method forimproving the plasma half-life of a Factor VIII molecule afterintravenous administration relative to human wild-type Factor VIII,comprising inactivating the proteolytic cleavage site between Arg1648and Glu1649, and, if present in the FVIII molecule, the proteolyticcleavage site between Arg1313 and Ala1314.

In another aspect, the present invention relates to a method forprolonging the time period for the thrombin peak level as determined ina thrombin generation assay over time in hemophilia A mice to fall below50 nM after intravenous administration of a Factor VIII moleculerelative to human wild type Factor VIII, comprising inactivating theproteolytic cleavage site between Arg1648 and Glu1649, and, if presentin the FVIII molecule, the proteolytic cleavage site between Arg1313 andAla1314.

In another aspect, the present invention relates to a method forretaining a higher activity for a Factor VIII molecule as determined bya one-stage FVIII:C assay after having been incubated for 4 days inhuman plasma at 37° C. relative to human wild type Factor VIII afterhaving been incubated for 4 days in human plasma at 37° C., comprisinginactivating the proteolytic cleavage site between Arg1648 and Glu1649,and, if present in the FVIII molecule, the proteolytic cleavage sitebetween Arg1313 and Ala1314.

A preferred embodiment of the methods described above are methodswherein a first amino acid selected from the amino acids at positions741 to 1647 of the Factor VIII sequence is fused with a second aminoacid selected from the amino acids at positions 1649 to 1690 of theFactor VIII sequence, whereby the proteolytic cleavage site betweenArg1648 and Glu1649, and, if present in the FVIII molecule, theproteolytic cleavage site between Arg1313 and Ala1314 is inactivated.

The preferred embodiments of the different aspects are applicablemutatis mutandis.

In yet another aspect, the present invention relates to a pharmaceuticalpreparation comprising a single chain Factor VIII molecule for use inthe treatment or prophylaxis of a bleeding disorder, preferablyhemophilia A, by (i) on the one hand non-intravenous administration,wherein the bioavailability of said single chain Factor VIII molecule isincreased by at least 25% as compared to human wild type Factor VIII oras compared to a B-domain deleted human Factor VIII molecule whereAsn745 is fused to Pro1640, administered at the same dose and in thesame manner, or (ii) on the other hand by intravenous administration,wherein (a) the plasma half-life of said single chain Factor VIIImolecule after intravenous administration is increased by at least 40%,relative to human wild type Factor VIII, administered at the same doseand in the same manner, or (b) the single chain Factor VIII moleculeexhibits a time period prolonged by at least 10 hours for the thrombinpeak level as determined in a thrombin generation assay over time inhemophilia A mice to fall below 50 nM after intravenous administrationrelative to human wild type Factor VIII, administered at the same doseand in the same manner.

In yet another aspect, the present invention relates to a pharmaceuticalpreparation comprising a single chain Factor VIII molecule for use inthe treatment or prophylaxis of a bleeding disorder, preferablyhemophilia A, wherein the single chain Factor VIII molecule retains atleast a 10% higher activity as determined by a one-stage FVIII:C assayafter having been incubated for 4 days in human plasma at 37° C.relative to human wild type Factor VIII after having been incubated for4 days in human plasma at 37° C.

In yet another aspect, the present invention relates to a pharmaceuticalpreparation comprising a single chain Factor VIII molecule for use inthe treatment or prophylaxis of a bleeding disorder, preferablyhemophilia A, by non-intravenous administration, wherein the dose ofsaid FVIII molecule can be decreased by at least 25% as compared to thatof a B-domain deleted Factor VIII molecule where Asn745 is fused toPro1640, administered at the same dose and in the same manner to achievethe same hemostatic activity in blood.

In another aspect, the present invention relates to the use of a singlechain Factor VIII molecule for achieving an increased stability afterreconstitution or a longer shelf life of a pharmaceutical preparationfor treating a bleeding disorder, wherein (i) the Factor VIII activityof the pharmaceutical preparation comprising the single chain FactorVIII molecule, after reconstitution and storage at room temperature for7 days after reconstitution is at least 10% higher than that of apharmaceutical preparation comprising the same amount of a B-domaindeleted Factor VIII molecule where Asn745 is fused to Pro1640, or (ii)wherein the single chain Factor VIII molecule retains at least a 10%higher activity as determined by a one-stage FVIII:C assay whenincubated for 4 days in human plasma at 37° C. relative to human wildtype Factor VIII after having been incubated for 4 days in human plasmaat 37° C. at the same concentration.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the results of Example 1. Various Factor VIII moleculeshave been provided as aqueous solutions, and their stability has beenmonitored over a time period of seven days. The loss in activity afterseven days of storage is much less for the single chain Factor VIIImolecule as compared to heterodimeric (two-chain) full length FactorVIII molecules (Beriate® and Helixate®) and to heterodimeric (two-chain)B-domain deleted constructs (ReFacto®).

FIG. 2 depicts the results of Example 2. Various lyophilized Factor VIIIpreparations were reconstituted to aqueous solutions, and theirstability has been monitored over a time period of seven days. The lossin activity after seven days of storage is much less for the singlechain Factor VIII molecule as compared to a heterodimeric (two-chain)full length Factor VIII molecule (Advate®) and to a heterodimeric(two-chain) and B-domain deleted construct (ReFacto®).

FIG. 3 depicts the results of Example 3. Three different Factor VIIImolecules have been injected subcutaneously in mice and theirbioavailability has been determined as described in Example 2. Thebioavailability of the single chain Factor VIII molecule issubstantially higher than that of a two chain and full length FactorVIII (Advate®) or a heterodimeric (two chain) B-domain deleted construct(ReFacto®).

FIG. 4 depicts the results of Example 4. The Factor VIII molecule of theinvention and 2 commercially obtainable FVIII preparations wereincubated at 37° C. after purification, lyophilization andreconstitution. The FVIII—samples were incubated at 37° C. for varyingtime periods (0, 0.25, 1, 2, 4 and 8 days) and the FVIII:C activity wasdetermined by an one-stage-coagulation assay. The values shown representthe average and standard deviation of two samples (except 0.25 days onlyone sample).

FIG. 5 depicts part of the results of Example 5. The pharmacokinetic(PK) profiles of scFVIII and full-length rFVIII (Advate®, BaxterHealthcare) were determined following a single I.V. injection tocynomolgus monkeys at a dose of 250 IU/kg.

FIG. 6 depicts part of the results of Example 5. The pharmacokinetic(PK) profiles of full-length rFVIII (Advate®, Baxter Healthcare) weredetermined following a single I.V. injection to hemophilia A mice at adose of 100 IU/kg.

FIG. 7 depicts part of the results of Example 6. The average peakthrombin levels from days 1-8 were determined after scFVIII orfull-length rFVIII (Advate®, Baxter Healthcare) were administered tohemophilia A mice at a dose of 250 IU/kg.

FIG. 8 depicts the results of Example 7. The pharmacokinetic (PK)profiles of full-length rFVIII (Advate®, Baxter Healthcare) and of aB-domain deleted Factor VIII (ReFacto®, Pfizer) was determined followinga single I.V. injection to VWF deficient mice at a dose of 100 IU/kg.

DETAILED DESCRIPTION

The present invention relates to a method for increasing the stabilityof a Factor VIII molecule after purification, lyophilization andreconstitution, comprising preventing proteolytic cleavage of the FactorVIII molecule into a first fragment comprising essentially the A1 domainand the A2 domain and a second fragment comprising essentially the A3domain, the C1 domain and the C2 domain.

This invention further pertains to a method for increasing the stabilityof a Factor VIII molecule after purification, lyophilization andreconstitution, comprising inactivating the proteolytic cleavage sitebetween Arg1648 and Glu1649 and, optionally inactivating the proteolyticcleavage site between Arg1313 and Ala1314, if present in the Factor VIIImolecule.

Factor VIII

The terms “blood coagulation Factor VIII”, “Factor VIII” and FVIII″ areused interchangeably herein. Mature human Factor VIII consists of 2332amino acids which are arranged in the following domain structure:

A1 a1 A2 a2 B a3 A3 C1 C2 A1: residues 1-336, A2: residues 373-710, B:residues 741-1648, A3. residues 1690-2019, C1: residues 2020-2172, andC2: residues 2173-2332.

In addition, there are three acidic regions a1 (337-372), a2 (711-740),and a3 (1649-1689). It is known that the acidic region a3 is involved inthe binding of the Factor VIII molecule to von Willebrand Factor (vWF)which plays an important role in blood coagulation. During secretion,the FVIII is cleaved between the B-domain and the a3 acidic region,resulting in a heterodimeric polypeptide The factor VIII heterodimerconsists of a light chain (comprising A3, C1 and C2) and a variablysized heavy chain (comprising A1, A2 and B). The latter is heterogeneousdue to limited proteolysis within the B-domain. In case of heterodimericB-domain deleted constructs the “heavy chain” comprises A1 and A2 butlacks part or all of the B-domain.

The amino acid sequence of the mature wild type form of human bloodcoagulation Factor VIII is shown in SEQ ID NO:2. The reference to anamino acid position of a specific sequence means the position of saidamino acid in the FVIII wild-type protein and does not exclude thepresence of mutations, e.g. deletions, insertions and/or substitutionsat other positions in the sequence referred to. For example, a mutationin “Glu2004” referring to SEQ ID NO:2 does not exclude that in themodified homologue one or more amino acids at positions 1 through 2332of SEQ ID NO:2 are missing. A DNA sequence encoding SEQ ID NO:2 is shownin SEQ ID NO:1.

“Blood coagulation Factor VIII” includes wild type blood coagulationFactor VIII as well as derivatives of wild type blood coagulation FactorVIII having the procoagulant activity of wild type blood coagulationFactor VIII. Derivatives may have deletions, insertions and/or additionscompared with the amino acid sequence of wild type Factor VIII.Preferred derivatives are FVIII molecules in which all or part of theB-domain has been deleted. Amino acid positions indicated throughoutthis application always refer to the position of the respective aminoacid in the full length mature (i.e. after signal peptide cleavage)wild-type FVIII.

The term “factor VIII” includes any factor VIII variants or mutantshaving at least 10%, preferably at least 25%, more preferably at least50%, most preferably at least 75% of the biological activity of wildtype factor VIII. A suitable test to determine the biological activityof Factor VIII is the one stage or the two stage coagulation assay(Rizza et al. 1982. Coagulation assay of FVIII:C and FIXa in Bloom ed.The Hemophilias. NY Churchchill Livingston 1992) or the chromogenicsubstrate FVIII:C assay (S. Rosen, 1984. Scand J Haematol 33: 139-145,suppl.). The content of these references is incorporated herein byreference.

As non-limiting examples, Factor VIII molecules include Factor VIIImutants preventing or reducing APC cleavage (Amano 1998. Thromb.Haemost. 79:557-563), albumin-fused FVIII molecules (WO 2011/020866 A2),FVIII-Fc fusion molecules (WO 04/101740 A), Factor VIII mutants furtherstabilizing the A2 domain (WO 97/40145), FVIII mutants resulting inincreased expression (Swaroop et al. 1997. JBC 272:24121-24124), FactorVIII mutants with reduced immunogenicity (Lollar 1999. Thromb. Haemost.82:505-508), FVIII reconstituted from differently expressed heavy andlight chains (Oh et al. 1999. Exp. Mol. Med. 31:95-100), FVIII mutantsreducing binding to receptors leading to catabolism of FVIII like HSPG(heparan sulfate proteoglycans) and/or LRP (low density lipoproteinreceptor related protein) (Ananyeva et al. 2001. TCM, 11:251-257),disulfide bond-stabilized FVIII variants (Gale et al., 2006. J. Thromb.Hemost. 4:1315-1322), FVIII mutants with improved secretion properties(Miao et al., 2004. Blood 103:3412-3419), FVIII mutants with increasedcofactor specific activity (Wakabayashi et al., 2005. Biochemistry44:10298-304), FVIII mutants with improved biosynthesis and secretion,reduced ER chaperone interaction, improved ER-Golgi transport, increasedactivation or resistance to inactivation and improved half-life(summarized by Pipe 2004. Sem. Thromb. Hemost. 30:227-237), and FVIIImutants having a deletion of all or part of the B-domain (see, e.g., WO2004/067566 A1, WO 02/102850 A2, WO 00/24759 A1 and U.S. Pat. No.4,868,112). All of these factor VIII mutants and variants areincorporated herein by reference in their entirety.

The term “single-chain Factor VIII” refers to a Factor VIII moleculewhich has not been proteolytically cleaved into two chains (e.g. a heavychain and a light chain) during secretion from the cells expressing saidFVIII molecule and, accordingly, is present as a single polypeptidechain.

Preventing Cleavage

The method of the invention comprises preventing proteolytic cleavage ofthe Factor VIII molecule into a first fragment comprising essentiallythe A1 domain and the A2 domain and a second fragment comprisingessentially the A3 domain, the 01 domain and the C2 domain. The term“preventing proteolytic cleavage” includes partially preventingproteolytic cleavage and completely preventing proteolytic cleavage. Itfurther includes the embodiment “reducing proteolytic cleavage”. Inother words, “preventing proteolytic cleavage of the Factor VIIImolecule” does not require completely abolishing any proteolyticcleavage such that substantially 100% of the Factor VIII moleculesexpressed and secreted by the host cells are single chain molecules(though this embodiment is encompassed by the method of the invention).Usually, the proteolytic cleavage of the Factor VIII molecule isprevented in a manner such that at least 50%, preferably at least 60%,more preferably at least 70%, more preferably at least 80%, morepreferably at least 90%, most preferably at least 95% of the Factor VIIImolecules expressed and secreted by the host cells are single chainmolecules. The incomplete prevention of cleavage may, at least in part,be due to the fact that there can be some minor cleavage sites withinthe B domain which can lead to proteolytic cleavage of a small portionof the Factor VIII molecules even if the major cleavage sites (at R1313and R1648) are absent. This minor cleavage may or may not be preventedin accordance with this invention.

The first fragment comprises essentially the A1 domain and the A2 domainof Factor VIII. The first fragment may comprise the A1 domain and the A2domain, each domain having exactly the amino acid sequence indicatedabove. For example, the first fragment may comprise at least amino acids1 to 740 of the amino acid sequence of SEQ ID NO:2. Alternatively, thefirst fragment may comprise a variant of this sequence, having aminoacid deletions, substitutions and/or insertions which do notsubstantially affect the Factor VIII activity. The first fragment mayadditionally comprise an N-terminal part of the B domain of Factor VIII.

The second fragment comprises essentially the A3 domain, the C1 domainand the C2 domain. The second fragment may comprise the A3 domain, theC1 domain and the C2 domain, each domain having exactly the amino acidsequence indicated above. For example, the second fragment may compriseat least amino acids 1690 to 2332 of the amino acid sequence shown inSEQ ID NO:2. Alternatively, the second fragment may comprise a variantof this sequence, having amino acid deletions, substitutions and/orinsertions which do not substantially affect the Factor VIII activity.The second fragment may additionally comprise a C-terminal part of theacidic a3 region.

The method of the invention comprises preventing the proteolyticcleavage during secretion of the recombinantly expressed FVIII molecule,which would result in a heterodimeric (two-chain) polypeptide. That is,the method includes obtaining a single-chain Factor VIIII molecule. Thiscan be achieved in various ways, e.g. by inactivating the proteolyticcleavage sites involved in the intracellular processing of the mature,one-chain FVIII into the heterodimeric FVIII eventually secreted by thehost cells.

In one embodiment the step of inactivating the proteolytic cleavage sitebetween Arg1648 and Glu1649 comprises deleting one or more amino acidsforming the protease recognition sequence. The cleavage site afterresidue 1648 is a furin-type cleavage site. The recognition sequence forthe protease in the Factor VIII sequence is LKRHQR. Preferably, theinactivation step comprises deleting one, two, three, four, five or moreof these amino acid residues forming the recognition sequence.Preferably, the inactivation step comprises deleting at least one basicamino acid within the recognition sequence, more preferably, theinactivation step comprises deleting at least the arginine at position1648. Still more preferably, the inactivation step comprises deleting atleast amino acids 1643 to 1648 of the Factor VIII sequence. If therespective FVIII derivative comprises Arg1313, the inactivation stepcomprises also deleting at least the arginine at position Arg 1313.Still preferably is deleting at least amino acids 1313 to 1648 of theFactor VIII sequence to inactivate both cleavage sites after 1313 and1648, respectively.

Most preferably, the inactivation step comprises deleting at least theamino acid sequence from residues 800 to 1648 from the Factor VIIIsequence, e.g. the amino acid sequence from residues 741 to 1648 fromthe Factor VIII sequence. In another preferred embodiment, a first aminoacid selected from the amino acids at positions 741 to 1647 of theFactor VIII sequence is fused with a second amino acid selected from theamino acids at positions 1649 to 1690 of the Factor VIII sequence,whereby the proteolytic cleavage during secretion is prevented.Preferred deletions are as follows:

-   -   amino acid 740 is fused to amino acid 1650, whereby amino acids        741 to 1649 are deleted;    -   amino acid 740 is fused to amino acid 1690, whereby amino acids        741 to 1689 are deleted;    -   amino acid 740 is fused to amino acid 1669, whereby amino acids        741 to 1668 are deleted;    -   amino acid 743 is fused to amino acid 1650, whereby amino acids        744 to 1649 are deleted;    -   amino acid 764 is fused to amino acid 1650, whereby amino acids        765 to 1649 are deleted;    -   amino acid 764 is fused to amino acid 1653, whereby amino acids        765 to 1652 are deleted;    -   amino acid 764 is fused to amino acid 1656, whereby amino acids        765 to 1655 are deleted;    -   amino acid 745 is fused to amino acid 1650, whereby amino acids        746 to 1649 are deleted;    -   amino acid 745 is fused to amino acid 1653, whereby amino acids        746 to 1652 are deleted;    -   amino acid 745 is fused to amino acid 1656, whereby amino acids        746 to 1655 are deleted;    -   amino acid 757 is fused to amino acid 1650, whereby amino acids        758 to 1649 are deleted;    -   amino acid 757 is fused to amino acid 1653, whereby amino acids        758 to 1652 are deleted;    -   amino acid 757 is fused to amino acid 1656, whereby amino acids        758 to 1655 are deleted;    -   amino acid 793 is fused to amino acid 1649, whereby amino acids        794 to 1648 are deleted;    -   amino acid 793 is fused to amino acid 1690, whereby amino acids        794 to 1689 are deleted;    -   amino acid 747 is fused to amino acid 1649, whereby amino acids        748 to 1648 are deleted;    -   amino acid 751 is fused to amino acid 1649, whereby amino acids        752 to 1648 are deleted;    -   amino acid 776 is fused to amino acid 1649, whereby amino acids        777 to 1648 are deleted;    -   amino acid 770 is fused to amino acid 1667, whereby amino acids        771 to 1666 are deleted.

The molecules resulting from the deletion are usually obtained in theform of single chain Factor VIII molecules.

Preferred single chain FVIII molecules have a deletion of all or part ofthe B-domain and a deletion of all or a part of the acidic a3 region, sothat the cleavage site at Arg1648 (which is usually cleaved duringsecretion) is deleted. Single chain FVIII molecules are disclosed in,e.g., WO 2004/067566 A1; US 2002/132306 A1; Krishnan et al. (1991)European Journal of Biochemistry vol. 195, no. 3, pages 637-644;Herlitschka et al. (1998) Journal of Biotechnology, vol. 61, no. 3,pages 165-173; Donath et al. (1995) Biochem. J., vol. 312, pages 49-55.These single-chain Factor VIII molecules described in these referencesare incorporated herein by reference.

The fusions referred to above may be direct fusions or indirect fusions.In the latter case, the deleted amino acids are replaced by aheterologous spacer. This embodiment is described in more detailhereinafter. It is possible that the deleted amino acids are replacedwith a peptidic linker consisting of about 1 to about 500 amino acids,or about 2 to 250 amino acids, or about 3 to about 100 amino acids, orabout 4 to about 50 amino acids, or about 5 to about 10 amino acids. Thepeptidic linker should be flexible and not immunogenic (Robinson et al.;PNAS (1998), Vol 95, p 5929). The peptidic linkers may consist of Glypreceded N-terminally to said Gly by multimers of the amino acidsequence GlyGlySer or GlyGlySerSer or any combination thereof, in aspecific embodiment the peptidic linker consists of 80 to 120 aminoacids.

In an alternative embodiment, one or more amino acids which form theprotease recognition site at residue 1313 and 1648 may be substitutedwith another amino acid such that the cleavage does not occur. Forexample, the basic amino acids may be replaced with hydrophobic aminoacids.

Preparation of Single-Chain Factor VIII

The step of “preventing proteolytic cleavage” or “inactivating aproteolytic cleavage site” is carried out prior to the purification,lyophilisation and reconstitution of the Factor VIII. The step of“preventing proteolytic cleavage” or “inactivating a proteolyticcleavage site” is typically carried out during the preparation of theFactor VIII molecule. The method of the invention may include preventingthe proteolytic cleavage during expression of the Factor VIII molecule(in host cells), or inactivating the proteolytic cleavage site atArg1313 and/or Arg1648 during the preparation of the nucleic acidencoding the Factor VIII molecule.

These steps of “preventing proteolytic cleavage” or “inactivating aproteolytic cleavage site” may include removing, from a nucleic acidencoding Factor VIII, a portion encoding the proteolytic cleavage siteat Arg1313 and/or Arg1648, in accordance with the embodiments describedabove. This typically results in a nucleic acid encoding single chainFactor VIII. Generally, the method of the invention may further includeproviding a nucleic acid encoding the single-chain Factor VIII, e.g. inan expression plasmid or vector.

The nucleic acid, the expression vector or the expression plasmid maythen be introduced into host cells, preferably mammalian host cells, forexpression. The method of the invention may further comprise culturingthe host cells under suitable conditions such that the modified FactorVIII molecule, e.g. the single chain Factor VIII molecule, is expressed;and optionally recovering (e.g. purifying) the modified Factor VIIImolecule from the host cells or from the culture medium. Generally,techniques of manipulating the nucleic acid encoding Factor VIII, ofculturing mammalian cells to allow expression of the Factor VIII, and ofpurifying Factor VIII from the cell culture medium are known in the art.

It is preferred to purify the single chain Factor VIII molecule to 80%purity, more preferably ≥95% purity and particularly preferred is apharmaceutically pure state that is greater than 99.9% pure with respectto contaminating macromolecules, particularly other proteins or/andnucleic acids, and free of infectious and pyrogenic agents. Preferably,an isolated or purified modified Factor VIII molecule is substantiallyfree of other polypeptides.

The methods of the invention may further comprise the steps ofpurifying, lyophilizing, and reconstituting the single chain FactorVIII. The reconstitution is preferably carried out by using water, e.g.“water for injection”.

Stability

The Factor VIII molecules prepared in accordance with the presentinvention exhibit enhanced stability relative to full length Factor VIIIand/or relative to a B-domain deleted Factor VIII molecule where Asn745is fused to Pro1640 (i.e. a B-domain deleted Factor VIII moleculeconsisting essentially of amino acids 1-745 and 1640-2332 of SEQ IDNO:2).

As used herein, the term “stability” refers to stability in aqueoussolution, preferably to stability in aqueous solution afterreconstitution of a lyophilized Factor VIII preparation, e.g. by addingwater to the lyophilized Factor VIII preparation. Typically, thelyophilized Factor VIII preparation is reconstituted with “water forinjection”.

The stability in aqueous solution can be determined by providing theFactor VIII molecule in aqueous solution and incubating it for a certainperiod of time. In a preferred embodiment, the conditions fordetermining the storage stability of the Factor VIII molecule are asfollows:

The Factor VIII molecule is provided in aqueous solution having thefollowing composition:

L-histidine 25 mM NaCl 225 mM calcium chloride 4 mM Tweene ® 80 0.03%(w/w) sucrose 2% (w/w) D-mannitol 8% (w/w) pH 7.0.

This solution is referred to hereinafter as “Buffer A”. The initialFactor VIII activity in the aqueous solution is preferably between 100IU/ml and 1,500 IU/ml, preferably it is 100 IU/ml.

The so prepared Factor VIII solution can then be incubated at 25° C. forat least 24 hours, preferably for at least two days, more preferably forat least five days, most preferably for seven or eight days. After theincubation period the stability is determined by measuring the FactorVIII activity in the solution, preferably by using a chromogenicsubstrate assay (e.g. Coamatic® Factor VIII, Chromogenix). The lower theloss in activity relative to the initial activity, the higher is thestability of the Factor VIII molecule. Most preferably, the stability isdetermined as in Example 1 or 2 below.

According to the present invention the loss in Factor VIII activity ofthe single-chain Factor VIII after seven days of storage under theabove-identified conditions is less than 15%, preferably less than 12%,most preferably less than 10%.

Typically, the initial Factor VIII activity at the start of theincubation period (t=0) is normalized to 100%. The remaining Factor VIIIactivity after 24 hours of storage in Buffer A at 25° C. is preferablyat least 95% of the initial Factor VIII activity. The remaining FactorVIII activity after 48 hours of storage in Buffer A at 25° C. ispreferably at least 95% of the initial Factor VIII activity. Theremaining Factor VIII activity after 4 days of storage in Buffer A at25° C. is preferably at least 90%, more preferably at least 95% of theinitial Factor VIII activity. The remaining Factor VIII activity after 7days of storage in Buffer A at 25° C. is preferably at least 85%, morepreferably at least 90%, most preferably at least 95% of the initialFactor VIII activity. The remaining Factor VIII activity after 8 days ofstorage in Buffer A at 25° C. is preferably at least 85%, morepreferably at least 90%, most preferably at least 95% of the initialFactor VIII activity.

The remaining Factor VIII activity of the single chain Factor VIII isusually higher than that of two-chain Factor VIII molecules (assumingthat both molecules have been incubated under identical conditions forthe same period of time).

The term “human full length two-chain Factor VIII” is used hereininterchangeably with the term “human wild-type Factor VIII”.

In one embodiment, the remaining Factor VIII activity of the singlechain Factor VIII is higher than that of human full length two-chainFactor VIII. In another embodiment, the remaining Factor VIII activityof the single chain Factor VIII is higher than that of a B-domaindeleted Factor VIII molecule where Asn745 is fused to Pro1640 (i.e. aB-domain deleted Factor VIII molecule consisting essentially of aminoacids 1-745 and 1640-2332 of SEQ ID NO:2).

Preferably, the remaining Factor VIII activity of the single chainFactor VIII after 48 hours of storage in Buffer A at 25° C. exceeds theremaining Factor VIII activity of human full length two-chain FactorVIII by at least 4 percentage points. It is also preferred that theremaining Factor VIII activity of the single chain Factor VIII after 48hours of storage in Buffer A at 25° C. exceeds the remaining Factor VIIIactivity of a B-domain deleted Factor VIII molecule where Asn745 isfused to Pro1640 (i.e. a B-domain deleted Factor VIII moleculeconsisting essentially of amino acids 1-745 and 1640-2332 of SEQ IDNO:2) by at least 4 percentage points.

In another embodiment, the remaining Factor VIII activity of the singlechain Factor VIII after 4 days of storage in Buffer A at 25° C. exceedsthe remaining Factor VIII activity of human full length two-chain FactorVIII by at least 5 percentage points. It is also preferred that theremaining Factor VIII activity of the single chain Factor VIII after 4days of storage in Buffer A at 25° C. exceeds the remaining Factor VIIIactivity of a B-domain deleted Factor VIII molecule where Asn745 isfused to Pro1640 (i.e. a B-domain deleted Factor VIII moleculeconsisting essentially of amino acids 1-745 and 1640-2332 of SEQ IDNO:2) by at least 5 percentage points.

In another embodiment, the remaining Factor VIII activity of the singlechain Factor VIII after 7 days of storage in Buffer A at 25° C. exceedsthe remaining Factor VIII activity of human full length two-chain FactorVIII by at least 5, preferably by at least 10 percentage points. It isalso preferred that the remaining Factor VIII activity of the singlechain Factor VIII after 7 days of storage in Buffer A at 25° C. exceedsthe remaining Factor VIII activity of a B-domain deleted Factor VIIImolecule where Asn745 is fused to Pro1640 (i.e. a B-domain deletedFactor VIII molecule consisting essentially of amino acids 1-745 and1640-2332 of SEQ ID NO:2) by at least 5, preferably by at least 10percentage points.

In another embodiment, the remaining Factor VIII activity of the singlechain Factor VIII after 8 days of storage in Buffer A at 25° C. exceedsthe remaining Factor VIII activity of human full length two-chain FactorVIII by at least 5, preferably by at least 10 percentage points. It isalso preferred that the remaining Factor VIII activity of the singlechain Factor VIII after 8 days of storage in Buffer A at 25° C. exceedsthe remaining Factor VIII activity of a B-domain deleted Factor VIIImolecule where Asn745 is fused to Pro1640 (i.e. a B-domain deletedFactor VIII molecule consisting essentially of amino acids 1-745 and1640-2332 of SEQ ID NO:2) by at least 5, preferably by at least 10percentage points.

Alternatively to Buffer A other buffers may also be used like, forexample the buffer used in Example 2 of the present invention.

The preferred pH range for the buffers of the present invention is a pHrange from 5.5 to 9.0, preferably a pH range from 6.0 to 8.5 andespecially preferred a pH range from 6.5 to 8.0.

The activity of Factor VIII can be determined by a chromogenic orclotting assay, or any other bioassay. Preferably, the Factor VIIIactivity is determined as shown in Example 1 below.

Bioavailability

In another embodiment, the Factor VIII molecule stabilized in accordancewith the present invention exhibits improved bioavailability afternon-intravenous injection, as compared to two chain human wild typeFactor VIII or compared to two chain human B-domain deleted Factor VIII.The non-intravenous injection is preferably subcutaneous, transdermal orintramuscular injection. Most preferably, the non-intravenous injectionis subcutaneous injection.

The term “bioavailability”, as used herein, refers to the proportion ofan administered dose of a Factor VIII or a FVIII-related preparationthat can be detected in plasma at predetermined times until a final timepoint after subcutaneous, intravenous or intradermal administration.Typically, bioavailability is measured in test animals by administeringa dose of between 10 IU/kg and 1000 IU/kg of the preparation (e.g. 400IU/kg body weight); obtaining plasma samples at pre-determined timepoints after administration; and determining the content of the FactorVIII or Factor VIII-related polypeptides in the samples using one ormore of a chromogenic or clotting assay (or any bioassay), animmunoassay, or an equivalent thereof. The bioavailability is expressedas the area under the curve (AUC) of the concentration or activity ofthe coagulation factor in plasma on the y-axis and the time afteradministration on the x-axis until a predefined final time point afteradministration. Preferably, this predefined time point is 72 or 48 hoursafter administration. Most preferably, the bioavailability is determinedas shown in Example 3 herein below. Relative bioavailability of a testpreparation refers to the ratio between the AUC of the test preparation(here: single chain Factor VIII) and that of the reference preparation(e.g. full length recombinant two-chain Factor VIII or two-chainB-domain deleted Factor VIII) which is administered in the same dose andway (e.g. intravenous, subcutaneous or intradermal) as the testpreparation.

According to the present invention, the bioavailability of the singlechain Factor VIII after subcutaneous injection is higher than that ofthe two-chain human wild type Factor VIII or of two-chain human B-domaindeleted Factor VIII. Preferably, the bioavailability (AUC over 72 hoursafter subcutaneous injection) is increased by at least 10%, morepreferably by at least 25%, more preferably by at least 50%, mostpreferably by at least 75%, relative to wild type FVIII. In anotherembodiment, the bioavailability (AUC over 72 hours after subcutaneousinjection) is increased by at least 10%, more preferably by at least20%, more preferably by at least 30%, most preferably by at least 40%,relative to a B-domain deleted Factor VIII molecule where Asn745 isfused to Pro1640 (i.e. a B-domain deleted Factor VIII moleculeconsisting essentially of amino acids 1-745 and 1640-2332 of SEQ IDNO:2).

Improvement of Plasma Half-Life (In-Vivo)

In another embodiment, the Factor VIII molecule stabilized in accordancewith the present invention exhibit increased pharmacokinetic (PK)parameters.

Factor VIII molecules of the invention can be tested by i.v. injectioninto different species like hemophilia A mice or cynomolgus monkeys e.g.at a dose of 100 IU/kg or 250 IU/kg respectively e.g. as determined in achromogenic assay. Blood samples are drawn at various time points afteradministration e.g. until 72 hours (hrs) in hemophilia A mice and e.g.until 24 hrs in cynomolgus monkeys. Citrate plasma is preparedimmediately and used for quantification of FVIII:C e.g. by a chromogenicassay system (FVIII:C) (Chromogenix-Instrumentation Laboratory SpA,Milan, Italy).

The AUC of the FVIII levels in plasma is calculated using the lineartrapezoidal rule to calculate AUC_(last): from t=0 to last observation.Terminal half-life (t_(1/2β)) is determined by a log-linear regressionusing the points of the terminal phase selected by the adjusted R2criterion. AUC: from t=0 to infinity (extrapolated by using theregression model of the terminal phase).

The single chain FVIII molecules according to the invention show atleast a 40%, preferably at least a 50%, even more preferably at least a60% increased terminal half life as compared to the terminal half-life ahuman wild-type Factor VIII administered at the same dose and in thesame manner.

Preferably the plasma half-life is determined as shown in Example 5.

Prolongation of Efficacy as Determined in a Thrombin Generation Assay(In-Vivo)

In another embodiment, the Factor VIII molecule stabilized in accordancewith the present invention exhibit a longer time period for the thrombinpeak level as determined in a thrombin generation assay over time inhemophilia A mice to fall below 50 nM after intravenous administrationrelative to human wild type Factor VIII. This test show that also thefunctionality of FVIII is stabilized in the molecules according to theinvention.

FVIII molecules according to the invention can be tested by firstadministering the FVIII molecule of the invention at an equimolar dose(e.g. at 250 IU/kg) intravenously into hemophilia A mice. At differenttime points (e.g. daily from day 1 to 8) citrated blood is collected anda thrombin generation assay (TGA) is performed e.g. by calibratedthrombinography (CAT) (Thrombinoscope, Netherlands) after intrinsicactivation in presence of Phospholipid (e.g. Rossix, MöIndal,Sweden)/Pathromtin® SL (Siemens Healthcare Diagnostics Products GmbH,Marburg, Germany) (1:30). Thrombin peak levels are recorded. The averageAUC of peak thrombin levels from days 1-8 is calculated by the lineartrapezoidal rule. The AUC of the two Factor VIII products are comparedusing an approximate F-test for the difference in AUC in a linear modelwith variable variances per time-point and treatment group resulting ina estimated time until peak levels of thrombin drop below a definedlimit ranging of 50 nM.

Preferably the efficacy in a thrombin generation assay is determined asshown in Example 6.

In hemophilia A mice scFVIII shows a favorable hemostatic activitycompared to human wild-type Factor VIII. This translates into anaveraged at least 10 hrs longer, preferably at least 15 hours longer andeven more preferred at least 20 hours longer thrombin generationactivity value for scFVIII versus full-length rFVIII before the thrombinpeak level falls below a level of 50 nM.

Retaining Higher FVIII:C Activity in Plasma (Ex Vivo)

In another embodiment, the Factor VIII molecule stabilized in accordancewith the present invention retain a higher activity as determined by aone-stage FVIII:C assay after having been incubated for 4 days in humanplasma at 37° C. relative to human wild type Factor VIII after havingbeen incubated for 4 days in human plasma at 37° C.; preferably whereinthe retained activity of the Factor VIII is at least 10% higher relativeto that of a human wild type Factor VIII after having been incubated for4 days in human plasma at 37° C.

Samples with Factor VIII molecules according to the invention can betested by diluting them into with FVIII deficient plasma (e.g. fromSiemens Healthcare Diagnostics) to 1 IU/mL FVIII:C (based on valuesdetermined by the chromogenic substrate assay). The FVIII—samples arethen incubated at 37° C. for varying time periods (e.g. for 0, 0.25, 1,2, 4 and 8 days) in presence of 0.05% Na-azide. After each incubationperiod, FVIII:C is then determined by a one-stage-coagulation assay e.g.by using Pathromtin-SL (Siemens Healthcare Diagnostics) as activator,normalized to the value at t=0 (% FVIII:C) and plotted versus theincubation time.

After a 4 day incubation at 37° C. the Factor VIII molecule of theinvention has retained at least a 10% higher FVIII:C activity,preferably at least 15% higher FVIII:C activity, preferably at least a20% higher FVIII:C activity, preferably at least a 25% higher FVIII:Cactivity, preferably at least a 30% higher FVIII:C activity.

Preferably the activity in plasma is determined as shown in Example 4.

Treatment and Prophylaxis

The single-chain Factor VIII constructs in accordance with the presentinvention having increased stability after reconstitution can beadministered in the treatment or prophylaxis of bleeding disorders.

As used herein, the term “bleeding disorders” includes familial andacquired hemophilia A and B, familial or acquired von Willebranddisease, familial or acquired deficiency of any coagulation factor, alltypes of trauma, blunt or penetrating, leading to severe hemorrhageeither from a single organ, a bone fraction or from polytrauma, bleedingduring surgical procedures including peri- or postoperative haemorrhage,bleeding due to cardiac surgery including patients undergoingextracorporal circulation and hemodilution in pediatric cardiac surgery,intracerebral hemorrhage, subarachnoid hemorrhage, sub- or epiduralbleeding, bleedings due to blood loss and hemodilution, by non-plasmaticvolume substitution leading to reduced levels of coagulation factors inaffected patients, bleedings due to disseminated intravascularcoagulation (DIC) and a consumption coagulopathy, thrombocytedysfunctions, depletion and coagulopathies, bleeding due to livercirrhosis, liver dysfunction and fulminant liver failure, liver biopsyin patients with liver disease, bleeding after liver and other organtransplantations, bleeding from gastric varices and peptic ulcerbleeding, gynaecological bleedings as dysfunctional uterine bleeding(DUB), premature detachment of the placenta, periventricular haemorrhagein low birth weight children, post partum haemorrhage, fatal distress ofnewborns, bleeding associated with burns, bleeding associated withamyloidosis, hematopoietic stem cell transplantation associated withplatelet disorder, bleedings associated with malignancies, infectionswith haemorrhaging viruses, bleeding associated with pancreatitis.

The components of the pharmaceutical preparation may be dissolved inconventional physiologically compatible aqueous buffer solutions towhich there may be added, optionally, pharmaceutical excipients toprovide the pharmaceutical preparation. The components of thepharmaceutical preparation may already contain all necessarypharmaceutical, physiologically compatible excipients and may bedissolved in water for injection to provide the pharmaceuticalpreparation.

Such pharmaceutical carriers and excipients as well as the preparationof suitable pharmaceutical formulations are well known in the art (seefor example “Pharmaceutical Formulation Development of Peptides andProteins”, Frokjaer et al., Taylor & Francis (2000) or “Handbook ofPharmaceutical Excipients”, 3^(rd) edition, Kibbe et al., PharmaceuticalPress (2000)). In certain embodiments, a pharmaceutical composition cancomprise at least one additive such as a bulking agent, buffer, orstabilizer. Standard pharmaceutical formulation techniques are wellknown to persons skilled in the art (see, e.g., 2005 Physicians' DeskReference®, Thomson Healthcare: Montvale, N.J., 2004; Remington: TheScience and Practice of Pharmacy, 20th ed., Gennaro et al., Eds.Lippincott Williams & Wilkins: Philadelphia, Pa., 2000). Suitablepharmaceutical additives include, e.g., sugars like mannitol, sorbitol,lactose, sucrose, trehalose, or others, amino acids like histidine,arginine, lysine, glycine, alanine, leucine, serine, threonine, glutamicacid, aspartic acid, glutamine, asparagine, phenylalanine, or others,additives to achieve isotonic conditions like sodium chloride or othersalts, stabilizers like Polysorbate 80, Polysorbate 20, Polyethyleneglycol, propylene glycol, calcium chloride, or others, physiological pHbuffering agents like Tris(hydroxymethyl)aminomethan, and the like. Incertain embodiments, the pharmaceutical compositions may contain pHbuffering reagents and wetting or emulsifying agents. In furtherembodiments, the compositions may contain preservatives or stabilizers.In particular, the pharmaceutical preparation comprising the bloodcoagulation factor may be formulated in lyophilized or stable solubleform. The blood coagulation factor may be lyophilized by a variety ofprocedures known in the art. Lyophilized formulations are reconstitutedprior to use by the addition of one or more pharmaceutically acceptablediluents such as sterile water for injection or sterile physiologicalsaline solution or a suitable buffer solution.

The composition(s) contained in the pharmaceutical preparation of theinvention may be delivered to the individual by any pharmaceuticallysuitable means. Various delivery systems are known and can be used toadminister the composition by any convenient route. Preferably, thecomposition(s) contained in the pharmaceutical preparation of theinvention are delivered to the individual by non-intravenous injection.More preferably, the composition(s) of the invention are formulated forsubcutaneous, intramuscular, intraperitoneal, intracerebral,intrapulmonar, intranasal, intradermal or transdermal administration,most preferably for subcutaneous, intramuscular or transdermaladministration according to conventional methods. The formulations canbe administered continuously by infusion or by bolus injection. Someformulations may encompass slow release systems.

The composition(s) of the pharmaceutical preparation of the presentinvention is/are administered to patients in a therapeutically effectivedose, meaning a dose that is sufficient to produce the desired effects,preventing or lessening the severity or spread of the condition orindication being treated without reaching a dose which producesintolerable adverse side effects. The exact dose depends on many factorsas e.g. the indication, formulation, mode of administration and has tobe determined in preclinical and clinical trials for each respectiveindication.

In one embodiment of the invention, the plasma level of the coagulationfactor in the treated subject is, during a period from 5 hours afterinjection to 8 hours after non-intravenous injection, continuouslyhigher than 2%, preferably higher than 5%, more preferably higher than8%, most preferably higher than 10%, of the normal plasma level of thecoagulation factor in healthy subjects. The plasma level is to bedetermined as shown hereinafter in Example 3.

In one embodiment of the invention, the plasma level of the coagulationfactor in the treated subject is, during a period from 4 hours afterinjection to 16 hours after non-intravenous injection, continuouslyhigher than 2%, preferably higher than 5%, more preferably higher than8%, most preferably higher than 10%, of the normal plasma level of thecoagulation factor in healthy subjects.

In another embodiment of the invention, the plasma level of thecoagulation factor in the treated subject is, during a period from 3hours after injection to 24 hours after non-intravenous injection,continuously higher than 2%, preferably higher than 4%, more preferablyhigher than 6%, most preferably higher than 8%, of the normal plasmalevel of the coagulation factor in healthy subjects.

In another embodiment of the invention, the plasma level of thecoagulation factor in the treated subject is, during a period from 2hours after injection to 32 hours after non-intravenous injection,continuously higher than 2%, preferably higher than 3%, more preferablyhigher than 4%, most preferably higher than 5%, of the normal plasmalevel of the coagulation factor in healthy subjects.

Preferably, the dose of single-chain Factor VIII for one non-intravenousinjection is less than 1,000 IU/kg body weight, or less than 800 IU/kgbody weight, or less than 600 IU/kg body weight, or less than 400 IU/kgbody weight, e.g. at a dose of from about 10 IU/kg body weight to about1,000 IU/kg body weight, or from about 20 IU/kg body weight to about 800IU/kg body weight, or from about 30 IU/kg body weight to about 700 IU/kgbody weight, or from about 40 IU/kg body weight to about 600 IU/kg bodyweight, or from about 50 IU/kg body weight to about 500 IU/kg bodyweight, or from about 75 IU/kg body weight to about 400 IU/kg bodyweight, or from about 100 IU/kg body weight to about 300 IU/kg bodyweight, or from about 50 IU/kg body weight to about 1,000 IU/kg bodyweight, or from about 50 IU/kg body weight to about 800 IU/kg bodyweight, or from about 50 IU/kg body weight to about 700 IU/kg bodyweight, or from about 50 IU/kg body weight to about 600 IU/kg bodyweight, or from about 50 IU/kg body weight to about 500 IU/kg bodyweight, or from about 50 IU/kg body weight to about 400 IU/kg bodyweight, or from about 50 IU/kg body weight to about 300 IU/kg bodyweight, or about 50 IU/kg body weight to about 200 IU/kg body weight.The FVIII can be administered on its own, or as a complex with VWF.

The pharmaceutical composition(s) of the invention may be administeredalone or in conjunction with other therapeutic agents. These agents maybe incorporated as part of the same pharmaceutical.

EXAMPLES Example 1: Stability of Purified Factor VIII Molecules afterReconstitution

The following Factor VIII preparations were used in this Example:

Beriate®, a lyophilized human coagulation Factor VIII concentrate, wasobtained from CSL Behring GmbH. Beriate® comprises plasma-derived FactorVIII in heterodimeric form.

Helixate®, a lyophilized, recombinant coagulation Factor VIII wasobtained from CSL Behring GmbH. Helixate® contains recombinantlyproduced heterodimeric Factor VIII.

ReFacto® is a lyophilized Factor VIII preparation containingheterodimeric, B-domain-deleted Factor VIII produced by recombinanttechnology. It can be obtained from, e.g., Pfizer Pharma GmbH, Germany.

Beriate®, Helixate®, and ReFacto® are predominantly heterodimerictwo-chain polypeptides.

The construct termed “scFVIII” is a single-chain Factor VIII produced byrecombinant expression in mammalian cell culture cells. The single-chainFactor VIII used in this Example was obtained by directly fusing Asn764with Thr1653, and provided in lyophilized form after purification. Thatis, “scFVIII” is a single chain polypeptide consisting substantially ofamino acids 1-764 and 1653-2332 of SEQ ID NO:2.

Beriate®, Helixate®, and ReFacto® were reconstituted according themanufacturer's instructions as given in the package insert. “scFVIII”was reconstituted by dissolving the purified and lyophilized FVIIIpreparation in water for injection resulting in a composition containing25 mM L-histidine, 225 mM NaCl, 4 mM CaCl₂, 0.03% Tween 80, 2% sucrose,8% D-mannitol, pH 7.0.

The reconstituted FVIII products were incubated at 25° C. The FVIIIactivity of the products was determined in duplicates by a chromogenicsubstrate assay (Coamatic® Factor VIII, Chromogenix) at the followingtime points: 0 h, 6 h, 1 day, 2 days, 4 days, 7 days. Activity valueswere normalized to time point 0.

The results are shown in the following Table and in FIG. 1.

TABLE 1 Factor VIII activity over time Time (days) 0 d 0.25 d 1 d 2 d 4d 7 d Beriate ® 100.0 103.8 92.7 97.9 90.0 82.7 Helixate ® 100.0 101.4103.3 95.5 85.0 78.6 ReFacto ® 100.0 106.7 83.7 94.0 93.6 77.3 scFVIII100.0 107.8 99.4 102.0 99.0 93.3

As can be seen, “scFVIII” shows the lowest loss in activity and,consequently, is the most stable Factor VIII molecule.

Example 2: Stability of Purified Factor VIII Molecules afterReconstitution

The following Factor VIII preparations were used in this Example:

ReFacto® and “scFVIII” were the same as used in Example 1, with thedifference that “scFVIII”, provided by CSL Behring GmbH, was applied ina formulation containing different excipients. Advate® is a full-length,heterodimeric, recombinant Factor VIII preparation which was purchasedin lyophilized form from Baxter.

Advate® and ReFacto® were reconstituted according the manufacturer'sinstructions as given in the package insert. “scFVIII” was reconstitutedin water for injection resulting in a composition containing 20 mML-histidine, 280 mM NaCl, 3.4 mM CaCl₂, 0.02% Tween 80, 0.6% sucrose, pH7.0. The sample “scFVIII 001” had an initial FVIII activity of 100IU/ml, the sample “scFVIII 0006” had an initial FVIII activity of 400IU/ml. The reconstituted FVIII products were incubated at 25° C. TheFVIII activity of the products was determined in duplicates by achromogenic substrate assay (Coamatic® Factor VIII, Chromogenix) at thefollowing time points: 0 h, 6 h, 1 day, 2 days, 4 days, 8 days. Activityvalues were normalized to time point 0.

The results are shown in the following Table and in FIG. 2.

TABLE 2 Factor VIII activity over time Time (days) 0 d 0.25 d 1 d 2 d 4d 8 d Advate 100.0 94.9 100.6 89.8 74.1 73.7 ReFacto 100.0 92.0 88.892.1 74.3 80.6 scFVIII 001 100.0 99.2 95.6 99.2 89.0 95.1 scFVIII 006100.0 96.8 97.6 100.0 88.9 92.3

As can be seen, “scFVIII” shows the lowest loss in activity and,consequently, is the most stable Factor VIII molecule.

Example 3: Bioavailability of Factor VIII Molecules

Advate®, ReFacto® and “scFVIII” were the same as used in Example 2 andreconstituted as described in Example 2.

Factor VIII knockout mice were used as animal model for hemophilia A.These mice lack exons 16 and 17 and thus do not express FVIII (Bi L. etal, Nature genetics, 1995, Vol 10(1), 119-121; Bi L. et al, Blood, 1996,Vol 88(9), 3446-3450). This allows the analysis of FVIII levelsfollowing treatment by quantification of FVIII activity in the plasma ofthe ko mice.

To assess whether extravascular injections might be an option for animproved therapy with human FVIII, subcutaneous injection was chosen.The design of the non-clinical pharmacokinetic study performed isdetailed in table 3 below. Plasma levels of Factor VIII activity weredetermined following a single subcutaneous injection of the respectiveFVIII preparation (detailed treatment groups in table 3) to a hemophiliaA model.

Corresponding groups were treated with the same dose of FVIII:chromogenactivity. For a single application the Factor VIII was provided in avolume of 200 μL (identical volumes for all groups) prior tosubcutaneous application to FVIII knockout (ko) mice weighing about 25g. The treatment groups are summarized in table 3.

Under short term anesthesia, blood samples were drawn, anticoagulatedusing sodium citrate to 10% citrate blood, processed to plasma andstored at −70° C. for the determination of FVIII activity. The samplingtime points are detailed in table 4. Quantification of FVIII activity inplasma was performed by a standard, aPTT based approach (BehringCoagulation Timer). The animals were kept at standard housingconditions.

TABLE 3 Treatment groups FVIII: chromogen/ Additive volume No. TreatmentDose [mL/kg] schedule route N 1 Advate ® 400 IU/kg 8 single injections.c. 25 (t = 0) 2 ReFacto ® 400 IU/kg 8 single injection s.c. 25 (t = 0)3 “scFVIII” 400 IU/kg 8 single injection s.c. 20 (t = 0)Results

The results are summarized in Table 4 and FIG. 3.

TABLE 4 timepoint scFVIII ReFacto ® Advate ® [hours] mean SD n mean SD nmean SD n 1 5.83 1.84 5 6.35 7.62 5 3.82 3.30 5 4 11.53 5.35 5 4.77 3.475 3.16 2.72 5 8 20.03 9.91 5 9.81 6.06 5 5.52 3.92 5 16 7.75 4.78 5 4.911.98 5 2.77 2.10 5 24 3.00 3.25 5 0.66 0.69 5 0.31 0.68 5 32 3.30 2.36 52.11 1.53 5 3.13 1.78 5 48 3.71 1.52 5 6.01 5.28 5 4.15 1.95 5 72 1.232.63 5 0.00 0.00 5 0.00 0.00 5 AUC_(0-72h) 383.9 275.1 195.1 (h × % ofthe norm)

Subcutaneous injection of 400 IU/kg single chain FVIII (“scFVIII”) toFVIII ko mice resulted in a significant increase of FVIII activity inplasma level as compared to administration of heterodimeric full lengthFVIII (Advate®) or heterodimeric B-domain-deleted FVIII (ReFacto®). Thatis, the single chain Factor VIII molecule shows the highest in vivobioavailability after subcutaneous injection into mice. The two chainfull length construct Advate®, as well as the two chain B-domain deletedpreparation ReFacto® showed substantially lower bioavailability.

Example 4: Stability of Factor VIII Molecules in Plasma (In-Vitro)

Different FVIII products (Advate®, ReFacto AF® and two lots of scFVIIIas used in Example 2) were diluted with FVIII deficient plasma (SiemensHealthcare Diagnostics) to 1 IU/mL FVIII:C (based on values determinedby the chromogenic substrate assay). The FVIII—samples were incubated at37° C. for varying time periods (0, 0.25, 1, 2, 4 and 8 days) inpresence of 0.05% Na-azide. After each incubation period, FVIII:C wasdetermined by one-stage-coagulation assay using Pathromtin-SL (SiemensHealthcare Diagnostics) as activator, normalized to the value at t=0 (%FVIII:C) and plotted versus the incubation time. The values shownrepresent the average and standard deviation of two samples (except 0.25days only one sample).

TABLE 5 Average % Activity Compared to Time 0 0 d 0.25 d 1d 2 d 4 d 8 dAdvate 100.00 90.63 81.25 79.69 70.31 59.38 ReFacto 100.00 92.31 86.1583.08 70.77 58.46 scFVIII 001 100.00 93.33 93.33 96.67 93.33 93.33scFVIII 006 100.00 96.88 96.88 93.75 93.75 90.63

Example 5: Stability of Factor VIII Molecules in Plasma (In-Vivo)

The pharmacokinetic (PK) profiles of scFVIII and full-length rFVIII(Advate®, Baxter Healthcare) was determined following a single I.V.injection to cynomolgus monkeys (FIG. 5 and Table 6) and hemophilia Amice (FIG. 6 and Table 7) at doses of 250 IU/kg and 100 IU/kg,respectively. Test items were dosed according to labeled activity forAdvate® and chromogenic activity (FVIII:C) for scFVIII. Blood sampleswere drawn predose (monkeys only) and at various time points afteradministration until 72 hours (hrs) in hemophilia A mice and until 24hrs in cynomolgus monkeys. Citrate plasma was prepared immediately andused for quantification of FVIII:C by a chromogenic assay system(FVIII:C) (Chromogenix-Instrumentation Laboratory SpA, Milan, Italy).

The AUC of the FVIII levels in plasma was calculated using the lineartrapezoidal rule to calculate AUC_(last): from t=0 to last observation.Terminal half-life (t_(1/2β)) was determined by a log-linear regressionusing the points of the terminal phase selected by the adjusted R2criterion. AUC: from t=0 to infinity (extrapolated by using theregression model of the terminal phase).

In cynomolgous monkeys scFVIII showed a ˜1.6 fold enhanced AUC_(0-tlast)or t_(1/2β) with a correspondingly ˜2 fold lower clearance (CL), whileFVIII activity peak levels (C_(max)), representative of in vivo recovery(IVR), and volume of distribution at steady state (V_(ss)) appeared moresimilar versus full-length rFVIII. These PK parameter results wereobtained from n=10 animals after toxicokinetic data from 8 additionalmonkeys, when dosed during the GLP—toxicity studies with 250 IU/kg ofscFVIII, were included (Table 6 and FIG. 5).

In hemophilia A mice enhancement of AUC_(0-tlast), of mean residencetime (MRT), time until 5% FVIII activity trough levels, terminalhalf-life and a correspondingly lower CL ranged between 1.6-2 fold forscFVIII, while C_(max), representative of IVR, and V_(ss) appearedsimilar versus full-length rFVIII. AUC_(0-tlast) and t_(1/2β) resultsobtained after rVIII-SingleChain treatment were significantly betterthan for full-length rFVIII with an AUC_(0-tlast) ratio of 1.97 (90%confidence interval (CI): 1.7-2.3; p-value(ratio=1):<0.0001), and at_(1/2β) ratio of 1.65 (90% CI: 1.11-2.70; p-value(ratio=1): 0.036(Table 7 and FIG. 6).

TABLE 6 PK parameters of scFVIII and full-length rFVIII in cynomolgusmonkeys scFVIII Full-length Parameters (n = 10) rFVIII (n = 2)AUC_(0-tlast) (hrs IU/mL) 78.4 49.1 C_(max) (IU/mL) 7.8 8.7 CL((mL/hrs)/kg) 2.1 4.7 t_(1/2β) (hrs) 11.0 6.8

TABLE 7 PK parameters of scFVIII and full-length rFVIII in hemophilia Amice Full-length Parameters scFVIII rFVIII AUC_(0-last) (hrs IU/mL) 3518 C_(max) (IU/mL) 2.3 2.2 CL ((mL/hrs)/kg) 2.7 5.5 MRT (hrs) 18 10V_(SS) (mL/kg) 50 57 t_(1/2β) (hrs) 15.9 9.7 Time until 0.05 IU/mL (hrs)73 39

Both sets of PK parameters reflect the increased stability of scFVIIIafter purification, lyophilization, reconstitution in vivo in plasmaafter administration to the two animal species tested.

Example 6: Thrombin Generation Assay in Hemophilia a Mice (Ex Vivo)

Citrate—(10% v/v) hemophilia A mouse blood was terminally collectedunder deep anesthesia at different time-points (days 1-8) when scFVIIIor full-length rFVIII (Advate®), were dosed @ a level of 250 IU/kg. TGAwas performed by calibrated thrombinography (CAT, Thrombinoscope,Netherlands) after intrinsic activation in presence of Phospholipid(Rossix, MöIndal, Sweden)/Pathromtin® SL (Siemens Healthcare DiagnosticsProducts GmbH, Marburg, Germany) (1:30). Thrombin peak levels wererecorded. The average AUC of peak thrombin levels from days 1-8 wascalculated by the linear trapezoidal rule. The AUC of the two FactorVIII products were compared using an approximate F-test for thedifference in AUC in a linear model with variable variances pertime-point and treatment group resulting in estimated time until peaklevels of thrombin drop below a defined limit ranging between 50-250 nM.

In hemophilia A mice scFVIII showed a favorable hemostatic activitycompared to full-length rFVIII as indicated by the estimated time untilpeak levels of thrombin drop below a defined limit ranging from 50-250nm peak level (FIG. 8 and Table 8). This translated into an averaged 20hrs longer thrombin generation activity value for scFVIII versusfull-length rFVIII for the thrombin peak level interval between 50 and250 nM. When assessing the area under the peak curve between days 1-8the thrombin generation activity of scFVIII was significantly betterwith p(AUC_(TGA Peak)-ratio=1)=0.0002 (estimated ratio 1.26, 90% CI:1.14-1.39) compared to full-length FVIII, or in other words it tooksignificantly longer for scFVIII to fall below a thrombin peak level of50 nm after administration than for the human wild-type Factor VIIIAdvate®.

These results again confirmed the increased functional stability ofscFVIII after purification, lyophilization and reconstitution.

TABLE 8 Peak nm Thrombin CSL627 (N = 8 − 14) Advate (N = 7 − 14) time[hrs] mean SD mean SD 0 0 0 0 0 24 325.5 40.2 343.4 40.41 32 294 102.8305.2 72.13 48 277.1 22.44 279.6 30.92 72 283.1 42.88 163.6 79.87 96115.4 29.63 75.44 35.28 120 91.85 57.63 55.17 30.89 144 45.6 36.15 24.6113.52 168 8.453 12.66 14.13 19.44 192 3.901 9.384 4.71 8.1

Example 6: Stability of Factor VIII Molecules in vWF Deficient Plasma(In-Vivo)

scFVIII was reconstituted in 2.5 mL water for injection. ReFacto AF® andAdvate® were reconstituted according to the description in the packageinsert. All test articles were aliquoted and stored immediately frozenat approximately −70° C. Prior to administration test articles werediluted with formulation buffer for CSL 627 to get a minimum practicalvolume ensuring a reliable administration.

12 VWF ko mice (6 female/6 male) per group received a single i.v.injection of 100 IU/kg of either scFVIII based on chromogenic FVIIIactivity and ReFacto AF® or Advate® based on the labeled FVIII activityinto the lateral tail vein. Following administration of the differenttest items blood samples were drawn for determination of FVIII plasmalevels at 0.083, 0.5, 1, 2, 4, 7, 16 and 24 hours from n=2-3 mice pertime point. Blood samples were processed to 10% citrate (3.13% w/v)plasma and subsequently subjected to FVIII plasma level analysis usingthe chromogenic assays system The chromogenic FVIII activity wasdetermined using the COAMATIC® FVIII test kit from Chromogenix, Italy.

The AUC of the FVIII levels in plasma was calculated using the lineartrapezoidal rule to calculate AUClast:from t=0 to last observation.

Likewise to results obtained after i.v. administration to FVIII ko miceand normal monkeys as well as s.c. administration to FVIII ko miceanalysis the exposure to CSL627 was higher compared to ReFacto AF® andAdvate®. Since analysis of the AUC, the most relevant and representativePK parameter for systemic exposure yielded a 30% higher AUC value afteradministration of CSL627 compared to both ReFacto AF® and Advate®.Again. these observations reflect the increased intrinsic stability ofscFVIII after purification, lyophilization, reconstitution in vivo inplasma after administration to mice lacking systemic, circulating VWF,hence in absence of its shielding and protective effect for systemic,circulating FVIII.

TABLE 9 Plasma levels of scFVIII compared to Advate ® and ReFacto AF ®after administration to VWF deficient mice at a dose level of 100 IU/kgCSL627 Advate ® ReFacto AF ® Baseline 0.0 0.0 0.0 Total Area 2427 16541714 Total Peak Area 2427 1654 1714 Number of Peaks 1.000 1.000 1.000Peak 1 First X = 0.0830 0.0830 0.0830 Last X = 24.00 24.00 24.00 Peak X= 0.0830 0.0830 0.0830 Peak Y = 1770 1131 1248 Area under curve = 24271654 1714 % Area = 100.0 100.0 100.0

The invention claimed is:
 1. A method of treatment or prophylaxis for ableeding disorder, comprising administering a modified Factor VIIImolecule to a subject, wherein the modified Factor VIII moleculecomprises a Factor VIII molecule and a peptidic linker, wherein theFactor VIII molecule is modified by inactivating the proteolyticcleavage site between Arg1648 and Glu1649, and, if present in the FactorVIII molecule, the proteolytic cleavage site between Arg1313 andAla1314, and wherein the modified Factor VIII molecule is administeredat a dosage of 20 IU/kg to 50 IU/kg, provides an equal or higher meanresidence time of the modified Factor VIII molecule as compared to meanresidence time of a higher dosage of an administered human wild-typeFactor VIII molecule.
 2. The method of claim 1, wherein the modifiedFactor VIII molecule and the wild-type Factor VIII molecule arerespectively administered intravenously.
 3. The method of claim 1,wherein the modified Factor VIII comprises SEQ ID NO: 2 modified to havea first amino acid selected from the amino acids at positions 741 to1647 of SEQ ID NO: 2 indirectly fused to a second amino acid selectedfrom the amino acids at positions 1649 to 1690 of SEQ ID NO: 2, deletingthe intervening amino acids and replacing them with a peptidic linker,whereby the proteolytic cleavage site between Arg1648 and Glu1649, and,if present in the modified Factor VIII molecule, the proteolyticcleavage site between Arg1313 and Ala1314, is inactivated.
 4. A methodof treatment or prophylaxis for a bleeding disorder, comprisingadministering a modified Factor VIII molecule to a subject, wherein themodified Factor VIII molecule comprises a Factor VIII molecule and apeptidic linker, wherein the Factor VIII molecule is modified byinactivating the proteolytic cleavage site between Arg1648 and Glu1649,and, if present in the Factor VIII molecule, the proteolytic cleavagesite between Arg1313 and Ala1314, and wherein the modified Factor VIIImolecule is administered at a dosage of 20 IU/kg to 50 IU/kg, and whilemaintaining or reducing the clearance rate of the modified Factor VIIImolecule as compared to the clearance rate of a higher dosage of anadministered human wild-type Factor VIII molecule.
 5. The method ofclaim 4, wherein the modified Factor VIII molecule and the wild-typeFactor VIII molecule are respectively administered intravenously.
 6. Themethod of claim 4, wherein the modified Factor VIII comprises SEQ ID NO:2 modified to have a first amino acid selected from the amino acids atpositions 741 to 1647 of SEQ ID NO: 2 indirectly fused to a second aminoacid selected from the amino acids at positions 1649 to 1690 of SEQ IDNO: 2, deleting the intervening amino acids and replacing them with apeptidic linker, whereby the proteolytic cleavage site between Arg1648and Glu1649, and, if present in the modified Factor VIII molecule, theproteolytic cleavage site between Arg1313 and Ala1314, is inactivated.7. A method of treatment or prophylaxis for a bleeding disorder,comprising administering a modified Factor VIII molecule to a subject,wherein the modified Factor VIII molecule comprises a Factor VIIImolecule and a peptidic linker, wherein the Factor VIII molecule ismodified by inactivating the proteolytic cleavage site between Arg1648and Glu1649, and, if present in the Factor VIII molecule, theproteolytic cleavage site between Arg1313 and Ala1314, and wherein themodified Factor VIII molecule is administered at a dosage of 20 IU/kg to50 IU/kg, while maintaining or increasing the area under the plasmaconcentration-time curve (AUC) of the modified Factor VIII molecule ascompared to the AUC of a higher dosage of an administered humanwild-type Factor VIII molecule.
 8. The method of claim 7, wherein themodified Factor VIII molecule and wild-type Factor VIII molecule arerespectively administered intravenously.
 9. The method of claim 7,wherein the modified Factor VIII comprises SEQ ID NO: 2 modified to havea first amino acid selected from the amino acids at positions 741 to1647 of SEQ ID NO: 2 indirectly fused to a second amino acid selectedfrom the amino acids at positions 1649 to 1690 of SEQ ID NO: 2, deletingthe intervening amino acids and replacing them with a peptidic linker,whereby the proteolytic cleavage site between Arg1648 and Glu1649, and,if present in the modified Factor VIII molecule, the proteolyticcleavage site between Arg1313 and Ala1314, is inactivated.
 10. A methodof treatment or prophylaxis for a bleeding disorder, comprisingadministering a modified Factor VIII molecule to a subject, wherein themodified Factor VIII molecule comprises a Factor VIII molecule and apeptidic linker, wherein the Factor VIII molecule is modified byinactivating the proteolytic cleavage site between Arg1648 and Glu1649,and, if present in the Factor VIII molecule, the proteolytic cleavagesite between Arg1313 and Ala1314, and wherein the modified Factor VIIImolecule is administered at a dosage of 20 IU/kg to 50 IU/kg, whilemaintaining or increasing the plasma half-life of the modified FactorVIII molecule as compared to the plasma half-life of a higher dosage ofan administered human wild-type Factor VIII molecule.
 11. The method ofclaim 10, wherein the modified Factor VIII molecule and wild-type FactorFVIII molecule are respectively administered intravenously.
 12. Themethod of claim 10, wherein the modified Factor VIII comprises SEQ IDNO: 2 modified to have a first amino acid selected from the amino acidsat positions 741 to 1647 of SEQ ID NO: 2 indirectly fused to a secondamino acid selected from the amino acids at positions 1649 to 1690 ofSEQ ID NO: 2, deleting the intervening amino acids and replacing themwith a peptidic linker, whereby the proteolytic cleavage site betweenArg1648 and Glu1649, and, if present in the modified Factor VIIImolecule, the proteolytic cleavage site between Arg1313 and Ala1314, isinactivated.
 13. The method of claim 2, wherein the dosage of themodified Factor VIII molecule is 25% lower than the higher dosage of anadministered human wild-type Factor VIII molecule.
 14. The method ofclaim 2, wherein the dosage of the modified Factor VIII molecule is 10%lower than the higher dosage of an administered human wild-type FactorVIII molecule.
 15. The method of claim 14, wherein the modified FactorVIII molecule is fused to an immunoglobulin Fc region.
 16. The method ofclaim 5, wherein the dosage of the modified Factor VIII molecule is 25%lower than the higher dosage of an administered human wild-type FactorVIII molecule.
 17. The method of claim 5, wherein the dosage of themodified Factor VIII molecule is 10% lower than the higher dosage of anadministered human wild-type Factor VIII molecule.
 18. The method ofclaim 17, wherein the modified Factor VIII molecule is fused to animmunoglobulin Fc region.
 19. The method of claim 8, wherein the dosageof the modified Factor VIII molecule is 25% lower than the higher dosageof an administered human wild-type Factor VIII molecule.
 20. The methodof claim 8, wherein the dosage of the modified Factor VIII molecule is10% lower than the higher dosage of an administered human wild-typeFactor VIII molecule.
 21. The method of claim 20, wherein the modifiedFactor VIII molecule is fused to an immunoglobulin Fc region.
 22. Themethod of claim 11, wherein the dosage of the modified Factor VIIImolecule is 25% lower than the higher dosage of an administered humanwild-type Factor VIII molecule.
 23. The method of claim 11, wherein thedosage of the modified Factor VIII molecule is 10% lower than the higherdosage of an administered human wild-type Factor VIII molecule.
 24. Themethod of claim 23, wherein the modified Factor VIII molecule is fusedto an immunoglobulin Fc region.